1. Field of the Invention
The present invention relates to a STAT-induced STAT inhibitor. More particularly, the present invention is concerned with a protein having the ability to inhibit the function of a STAT protein in a mammalian JAK/STAT signal transduction pathway and a DNA coding for the same. The protein of the present invention is induced by STAT3 or STAT6, and has the ability to inhibit tyrosine phosphorylation of gp130 and STAT3, and it comprises an SH2 domain. The protein of the present invention can be advantageously used for screening a novel substance having the capability to regulate cytokine activity. The present invention is also concerned with an antisense DNA and an antisense RNA which are capable of inhibiting the biosynthesis of the above-mentioned protein; a monoclonal antibody capable of binding to the above-mentioned protein; a DNA probe and an RNA probe which are capable of hybridizing to the above-mentioned DNA; a replicable recombinant DNA molecule comprising a replicable expression vector and, operably inserted therein, the above-mentioned DNA; and a cell of a microorganism or cell culture, transformed with the replicable recombinant DNA molecule. Furthermore, the transformant of the present invention can also be used for screening a novel substance having the capability to regulate cytokine activity.
2. Prior Art
Proteinaceous chemical substances which participate in intercellular signal transduction are called cytokines. Various substances, such as interferons, interleukins and colony-stimulating factors, have been identified as cytokines. Cytokines are glycoproteins, and it is generally known that receptors specific for cytokines are expressed on the surface of a target cell for the cytokines and physiological activities of the cytokines (such as regulation of cell proliferation and differentiation) are exerted only upon occurrence of binding between the cytokines and the receptor molecules.
Elucidation of an intercellular signal transduction pathway of a cytokine and clarification of a control mechanism thereof are very important. The elucidation and clarification may possibly lead to a development of a novel pharmaceutical or therapeutic method. The signal transduction mechanisms of cytokines have been elucidated in recent studies. Most of the cytokine receptors consist of two or three polypeptide chains, namely a ligand-specific receptor chain and a signal transducer that is commonly used by various cytokines {see FASEB J. 6, 3387-3396 (1992); and Cell 69, 1121-1132 (1992)}. The nature of this receptor system explains the functional redundancy of cytokines. For example, Takeda et al. {Molecular Medicine vol. 33, extra edition, Meneki (Immunity) 1996-97, ii} disclose that a JAK kinase (Janus kinase) having tyrosine phosphorylation activity is bound to an intracellular domain of a cytokine receptor. Binding of the cytokine (which is a ligand) to the receptor induces the dimerization of the receptor components and, as a consequence, two receptor-associated JAK kinases move toward each other and mutually activate each other. As a result, the two JAK kinases themselves and the cytokine receptor are phosphorylated.
In this situation, the STAT (signal transducer and activator of transcription) protein in the cell binds to the phosphorylated cytokine receptor, and it transmits a signal from the cytokine bound to the cell membrane via receptor to target genes in the cell nucleus. gp130 is a component protein of various receptors, such as IL-6 (interleukin-6) receptor, IL-11 (interleukin-11) receptor and LIF (leukemia inhibitory factor) receptor, and it is known that the binding of a cytokine to a receptor results in the activation of a JAK kinase, which in turn phosphorylates gp130. A STAT binds to the phosphorylated gp130 and signal transduction occurs consequently.
A STAT protein is a protein identified in recent studies, and six different types of STAT proteins (STAT1 to STAT6) are known in the art. Signal transduction from a STAT protein to a target gene in the cell nucleus takes place in the following manner. The binding of the above-mentioned ligand (namely a cytokine) to the receptor activates a JAK kinase, and the activated JAK kinase phosphorylates the tyrosine residue of the STAT protein. Next, the phosphorylated STAT protein forms a homodimer with the same type of STAT, or forms a heterodimer with a different type of STAT. The resultant STAT homodimer or heterodimer is capable of transducing the signals from the cytokines to the genes in the cell nucleus, and thus, the STAT is activated. The activated STAT translocates to the target gene and binds to a specific site on the gene, thereby initiating RNA synthesis, followed by biosynthesis of a new protein.
Homo- or heterodimerization of ligand-binding receptor components (i.e., a ligand-receptor complex, namely, a complex composed of two receptor molecules each binding a ligand thereto) stimulates a unique cytokine signalling cascade {see Cell 80, 213-223 (1995)}, i.e., the JAK/STAT pathway {see Science 264, 1415-1421 (1994); Nature 377, 591-594 (1995); and Cell 84, 331-334 (1996)}, which subsequently induces the activation of target genes. However, little is known about the genes (targets) of which the expression is directly induced by the STAT family proteins. One feature of cytokines is the transient expression of their activity, which suggests that a negative feedback regulation operates in the cytokine-signal transduction. Only a few proteins which can exert this negative feedback regulation are known in the art. Examples of known proteins include:
(i) an SH2-domain-containing phosphotyrosine phosphatase (SHP-1) which associates with the tyrosinephosphorylated IL-3 receptor xcex2-chain and with erythropoietin receptor (EPO-R) {see Cell 85, 15 (1996); Mol. Cell. Biol. 13, 7577-7586 (1993); and Cell 80, 729-738 (1995)}; and
(ii) a cytokine-inducible-SH2-containing protein (CIS) which binds to the STAT5-binding sites of EPO-R {see EMBO J. 14, 2816-2826 (1995); and ditto 15, 2425-2433 (1996)}.
As is apparent from the above, the target genes of the STAT family proteins, and the feedback mechanisms responsible for switching off the cytokine signals have not been fully elucidated.
Therefore, the present inventors made an attempt to isolate a protein encoded by a novel gene which regulates the STAT signals, and to elucidate a novel feedback mechanism responsible for switching off the cytokine signals.
It is an object of the present invention to identify a novel protein regulating the STAT signals, and to provide a method for utilizing the novel protein in the field of medicine. It is another object of the present invention to provide a protein encoded by the STAT target gene (that is, a gene whose expression is induced by the STAT) which is capable of inhibiting the STAT activity, and also to provide a method for utilizing the novel protein in the field of medicine.
The inventors of the present invention have made extensive and intensive studies with a view toward isolating a gene which regulates the STAT signals and analyzing the functions thereof. Particularly, the present inventors prepared a monoclonal antibody against the known, highly conserved sequence motif GTFLLRFS (SEQ ID NO: 5 (Gly-Thr-Phe-Leu-Leu-Arg-Phe-Ser in 3-letter abbreviation) found in an SH2 (src homology-2) domain which is important for the signal transduction of STAT1 to STAT6 (the above-mentioned sequence is a phosphotyrosine recognition site of the SH2 domain). A murine thymus cDNA library consisting of 3 million bacteriophage plaques was screened using the prepared monoclonal antibody, and about twenty unknown genes (excluding the known genes, such as the genes encoding STAT3 and the like) were isolated.
The whole sequences of five of the isolated genes were determined, and two genes were found to contain an SH2 domain. One of these genes was later identified as a gene encoding CIS protein (cytokine inducible SH2-containing protein). The other gene was a novel gene encoding a protein consisting of 212 amino acids, and having an SH2 domain at the C-terminal region and a domain consisting of eight contiguous serine residues at the N-terminal region. Further studies have revealed that this novel protein, in general, is induced by gp130-transduced signalling pathways. A northern blot analysis confirmed that the novel gene is not expressed in the cells transfected with a dominant-negative form of STAT3 gene which is incapable of being tyrosine phosphorylated by a JAK kinase (the cells were transfected with STAT3 gene in which a tyrosine at the 705th residue is substituted by phenylalanine). In addition, the amino acid sequence was deduced from the nucleotide sequence of the novel gene, and the amino acid sequence of SEQ ID NO: 2 was obtained. The present inventors named the protein encoded by the novel gene xe2x80x9cSIIS-1xe2x80x9d (abbreviation for xe2x80x9cSTAT-induced inhibitor of STAT function-1xe2x80x9d). From the analysis of SIIS-1 in factor-dependent cell lines, it has become apparent that SIIS-1 is also induced by STAT6, and the results of immunoblotting with anti-phosphotyrosine monoclonal antibody revealed that SIIS-1 has the ability to inhibit the tyrosine phosphorylation of gp130 and STAT3.
Therefore, it is a principal object of the present invention to provide a substantially pure protein having the ability to inhibit the function of a STAT protein in a mammalian JAK/STAT signal transduction pathway, wherein the protein has the following characteristics:
(1) the protein is induced by STAT3 or STAT6;
(2) the protein has the ability to inhibit tyrosine phosphorylation of gp130 and STAT3; and
(3) the protein comprises an SH2 domain.
It is another object of the present invention to provide a DNA coding for the above-mentioned protein, particularly, the DNA having the nucleotide sequence of SEQ ID NO: 1.
It is still another object of the present invention to provide a method for screening a substance which can be advantageously used as a pharmaceutical or a diagnostic reagent, which comprises contacting a sample with the above-mentioned protein, and assessing the activation or inhibition of the protein by the substance as a criterion.
Still a further object of the present invention is to provide an antisense DNA and an antisense RNA capable of inhibiting the biosynthesis of the above-mentioned protein, a monoclonal antibody capable of binding to the above-mentioned protein, or a DNA probe and an RNA probe capable of hybridizing to the above-mentioned DNA.
Still a further object of the present invention is to provide a replicable recombinant DNA molecule, comprising a replicable expression vector and, operably inserted therein, a gene encoding SIIS-1; and a cell of a microorganism or cell culture, transformed with the replicable recombinant DNA.
Still a further object of the present invention is to provide a method for screening a substance having the capability to regulate cytokine activity in which the transformant is used.
The foregoing and other objects, features and advantages of the present invention will be apparent to those skilled in the art from the following detailed description and the appended claims taken in connection with the accompanying sequence listing and drawings.
SEQ ID NO: 1 is the nucleotide sequence (SEQ ID NO: 2) of the novel STAT inhibitor protein of the present invention. SEQ ID NO: 2 is the amino acid sequence of the novel STAT inhibitor protein of the present invention.